Studies in a variety of tissues have rekindled interest in the possible second messenger role of calcium. Hormonal regulation of liver cell function as well as stimulation of neuronal tissue by membrane depolarization may involve calcium-dependent phosphorylation. Although the actions of calcium do not appear to be mediated by a universal biochemical mechanism, I propose to study the possibility that certain of the physiological effects of calcium may be mediated or modulated by protein phosphorylation. Two calcium-dependent protein kinases and endogenous substrate proteins have been detected in brain cytosol fractionated on DEAE-cellulose. One of the two enzymes is dependent on the presence of calmodulin, a calcium-binding protein. These protein kinases will be purified and their biochemical and functional properties analyzed. This would involve characterization of their molecular properties such a molecular weight, subunit composition and their kinetic properties such as interaction with calcium, calmodulin, ATP and protein substrates. These properties, in addition to their substrate specificity, will enable a comparison with other protein kinases. Regulation of these enzymes by autophosphorylation and by other protein kinases will be examined. Experiments will be initiated to study the function of these kinases in preparations of brain slices and synaptosomes. Changes in the state of phosphorylation of proteins resulting from stimulation and depression of neuronal activity will be analyzed to determine which proteins may be regulated by calcium-dependent protein phosphorylation. Numerous biochemical effects of calcium may be mediated in a variety of tissues via such a phosphorylation mechanism.